Transfer-RNAs (tRNA) are pivotal molecules of translation and their ability to accurately and efficiently decode genetic information depends on post-transcriptional modification of nucleotides in the critical anticodon stem loop (ASL). Deficiencies in these modifications cause hereditary human mitochondrial disease and modified nucleosides serve as sensitive diagnostics, such as human cancer markers. The long-term goal of this research project is to develop a detailed understanding of the biosynthesis of ASL modifications and their roles in cellular physiology, and to identify novel targets in these pathways for therapeutic intervention. This application specifically aims at elucidating biochemically and structurally the molecular mechanisms underlying the biosynthesis of the universal modified nucleoside threonylcarbamoyladenosine (t6A37). t6A37 is a complex, ancient modification of the ASL found in all tRNAs decoding ANN codons in all life forms. It is critical fo tRNA function by preventing ribosomal frame shifting and promoting cognate codon recognition, tRNA translocation and recognition by synthetases. We have recently discovered that four essential enzymes, TsaC, TsaB, TsaD and TsaE, are required and sufficient for t6A37 biosynthesis in bacteria. Because the four enzymes are essential in bacteria, and two of them are unique to the bacterial domain, the t6A37 pathway is a compelling potential target for the development of a new generation of anti- bacterial therapeutics. In the proposed work, the catalytic mechanism of the first and universal enzyme in the pathway, TsaC, will be elucidated using a multi-pronged approach comprising kinetic, biochemical, NMR and crystallographic methods. Simultaneously, the precise roles of other three proteins in the biosynthetic process and their concerted interactions with each other and with substrates will be elucidated using a combination of approaches, including transient kinetics experiments, tRNA binding experiments, NMR studies, and X-ray crystallography. This is a multi-institutional collaborative project that summons cross-disciplinary expertise and methodologies to elucidate the mechanistic and structural basis for t6A37 biosynthesis, and establish the experimental foundation for the development of the t6A37 pathway as an antibacterial target.